CN108611438A - Differentiate H9 and H10 subtype avian influenza virus duplex RT-PCR detection primers group, kit and its application simultaneously - Google Patents
Differentiate H9 and H10 subtype avian influenza virus duplex RT-PCR detection primers group, kit and its application simultaneously Download PDFInfo
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- CN108611438A CN108611438A CN201810272777.1A CN201810272777A CN108611438A CN 108611438 A CN108611438 A CN 108611438A CN 201810272777 A CN201810272777 A CN 201810272777A CN 108611438 A CN108611438 A CN 108611438A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Abstract
The invention belongs to avian viral detection technique fields, differentiating the double RT PCR detection primers group of H9 and H10 subtype avian influenza virus, kit and its application simultaneously the invention discloses a kind of.Inventor is by design for the specific primer of H9 and H10 hypotype AIV HA genes, it is combined by obtaining a best primer of expanding effect to the screening that different primers combine, it then proceedes to optimize its primer concentration, annealing temperature and proliferation time, finally sets up the method that can detect H9 and H10 hypotypes AIV simultaneously.Its specificity and sensitivity tests show that this method can only amplify H9 and H10 hypotype AIV HA genes, cannot detect that other hypotype AIV and the nucleic acid of common poultry cause of disease, most the low energy amount of detecting are 5 × 104Copy/μ L plasmid templates, in addition the testing result of clinical sample also indicate that it is consistent completely with virus purification result.
Description
Technical field
The invention belongs to avian viral detection technique fields, more particularly to one kind differentiating H9 and H10 subtype avian influenzas simultaneously
Viral duplex RT-PCR detection primer group, kit and its application.
Background technology
Avian influenza virus (Avian influenza virus, AIV) is that influenza A belongs to member in orthomyxovirus section.
According to its surface antigen haemagglutinin albumen (Hemagglutinin, HA) and neuraminidase protein (Neuraminidase, NA)
Difference AIV can be divided into 16 kinds of different HA hypotypes (H1-H16) NA hypotypes (N1-N9) different with 9 kinds.H9 hypotypes
AIV was found from 1966 in the U.S. for the first time, and global aviculture has been given to cause extremely serious economic loss at present.From
Since the finder's infection for the first time of Guangdong in 1998 H9N2 hypotype AIV cases, H9N2 types AIV infection occurrences in human life parts are also constantly being sent out
It is raw.H10 hypotypes AIV is mainly separated to virus in the duck of the chicken of north America region, turkey, wild bird and Southern African region, exists in recent years
Also there is the report for being separated to H10 subtype avian influenza strains in the environment and poultry swab samples of South China of China.Before
2004 and 2010, there are H10 hypotypes AIV infection occurrences in human life parts respectively in Egyptian and Australia.Since in December, 2013, in
3 people successively occur for state infect the cases of H10N8 subtype avian influenza virus simultaneously to cause wherein 2 death, this is in the world for the first time
Report H10 subtype influenza virus infection people and lethal case.The monitoring of the epidemiology of low pathogenicity bird flu in recent years
Middle discovery H9 and H10 subtype avian influenza virus mixed infection generally existings, research also show to infect the H10N8 subtype influenza diseases of people
The partial interior gene source of poison is in H9N2 hypotypes AIV.Therefore, H9 and H10 hypotypes AIV is to poultry farming and human health health
It is of great significance.
Since there are Testing and appraisal period longer disadvantages for classical Virus Isolation method, and common serology side
Rule needs between more standard positive serum and different subtype serum there are different degrees of cross reaction, have certain office
It is sex-limited, so detection avian influenza virus mainly uses molecular biology method at present.The molecular biology especially detection sides PCR
Method has many advantages, such as that easy to operate, specificity is good, sensibility is high, has been obtained during the monitoring and diagnosis of avian influenza virus wide
General application.Due to AIV mixed infections generally existing and there are similar clinical symptoms, it is difficult to reflecting to it promptly and accurately
It does not distinguish.
Application of the multiple PCR technique in bird flu diagnosis in recent years is more and more but mixed about H9 and H10 hypotypes AIV
The multi-PCR detection method for closing infection sees also report.
Being disclosed in the information of the background technology part, it is only intended to increase understanding of the overall background of the invention, without answering
It has been the prior art well known to persons skilled in the art when being considered as recognizing or imply that the information is constituted in any form.
Invention content
For overcome the deficiencies in the prior art, the present invention provides a kind of while differentiating H9 and H10 subtype avian influenza virus two
Weight RT-PCR detection primers group, kit and its application, establish a kind of double RT- that can detect H9 and H10 hypotypes AIV simultaneously
PCR detection method not only can fast and accurately differentiate two kinds of different subtype AIV, also be carried to infect the AIV pathogenic surveillances of people
For technical support, there is highly important public health meaning.
In order to solve the above technical problems, the present invention uses following technical scheme：
It is a kind of to differentiate that H9 and H10 subtype avian influenza virus duplex RT-PCR detection primer groups, including 2 pairs of specificity are drawn simultaneously
Object is primer pair H9-F and H9-R, primer pair H10-F and H10-R respectively, is respectively provided with such as SEQ ID No.1 to SEQ ID
Sequence shown in No.4.
It is a kind of to differentiate H9 and H10 subtype avian influenza virus duplex RT-PCR detection kits simultaneously, which is characterized in that packet
It includes：2 × PCR Mix, 12.5 μ L, H9 and H10 hypotypes AIV totally 2 μ L as template, the addition of specific primer H9-F and H9-R
Addition for 0.75 μ L, specific primer H10-F and H10-R is 1 μ L, and final volume is complemented to 25 μ L with RNA-free water.
Preferably, the specific primer H9-F and H9-R, specific primer H10-F and H10-R, are respectively provided with
The sequence as shown in SEQ ID No.1 to SEQ ID No.4.
Preferably, the specific primer H9-F and H9-R, specific primer H10-F and H10-R are in PCR reactants
A corresponding mole final concentration is respectively 0.75pmol/ μ L, 0.75pmol/ μ L, 1pmol/ μ L, 1pmol/ μ L in system.
The present invention also provides it is described while differentiate H9 and H10 subtype avian influenza virus duplex RT-PCR detection primer groups
Or differentiate that H9 and H10 subtype avian influenza virus duplex RT-PCR detection kits are differentiating H9 and H10 hypotype fowl while described
Application in influenza virus.
Compared with prior art, the present invention has the advantages that：
(1) present invention is directed to the specific primer of H9 and H10 hypotype AIV HA genes by design, by different primers
The screening of combination obtains the primer combination of expanding effect, then proceedes to its primer concentration, annealing temperature and proliferation time
It optimizes, finally sets up the method that can detect H9 and H10 hypotypes AIV simultaneously.Its specificity and sensitivity tests show this
Method can only amplify H9 and H10 hypotype AIV HA genes, cannot detect other hypotype AIV and the core of common poultry cause of disease
Acid, most the low energy amount of detecting are 5 × 104Copy/μ L plasmid templates, in addition the testing result of clinical sample also indicate that itself and virus
Separating resulting is consistent completely.
(2) present invention has been successfully established can detect H9 and H10 hypotype AIV duplex RT-PCR methods simultaneously, and this method has
High specificity and high sensitivity, conveniently and quickly the advantages that, a PCR reaction can determine the mixing sense of H9 and H10 hypotypes AIV
Dye and Simple infection situation, facilitate grass-roots unit to apply, and the rapid differential diagnosis for H9 and H10 hypotypes AIV provides effective skill
Art supports.
Description of the drawings
The specific test result figure of H9 and H10 hypotype AIV duplex RT-PCR methods are detected while Fig. 1 is the present invention；
Wherein, M.DNA Marker DL1000；1.H9+H10；2.H9+H10；3.H9N2；4.H9N6；5.H10N2；
6.H6N6；7.H1N7；8.H2N3；9.H3N2；10.H5N2；11.H7N9；12.H8N4；13.H10N3；14.H12N5；
15.H13N5；16.H14N1；17.H15N5；18.NDV；19.IBV；20.ARV；21.ILTV；22.MG；23.Negative
control；
The sensitivity tests result figure of H9 and H10 hypotype AIV duplex RT-PCR methods are detected while Fig. 2 is the present invention；
Wherein, M is DNAMarker DL1000；1 is 5 × 109copies/μL；2 be 5 × 108copies/μL；3 for 5 ×
107copies/μL；4 be 5 × 106copies/μL；5 be 5 × 105copies/μL；6 be 5 × 104copies/μL；7 for 5 ×
103copies/μL；8 be 5 × 102copies/μL；9 be 5 × 101copies/μL；10 be 10copies/ μ L；11 are
Negative control；
Detection H9 and H10 hypotype AIV duplex RT-PCRs methods are to clinical sample test result while Fig. 3 is the present invention
Figure；
Wherein, M.DNA Marker DL1000；7、10、14and 16.Positive products of H9subtype
AIVs；2.Positive products of H9and H10subtype AIVs；The others were negative
products。
Specific implementation mode
With reference to specific embodiment, further details of elaboration is made to the present invention, but embodiments of the present invention are not
It is confined to the range of embodiment expression.These embodiments are merely to illustrate the present invention, range and is not intended to limit the present invention.This
Outside, after reading present disclosure, those skilled in the art can various modifications may be made to the present invention, these equivalent variations are same
Sample falls within the appended claims limited range of the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.Institute in following embodiments
Material, reagent for using etc., are commercially available unless otherwise specified.Avian influenza strain used in embodiment
And other avian viral Reference Strains are preserved by Veterinary Institute of Guangxi Zhuang Autonomous Region.
Embodiment 1：Design of primers
The HA gene sequences of avian influenza virus H9 and H10 hypotype AIV are downloaded from GenBank databases with reference to pertinent literature
Each gene nucleotide series are analyzed and are compared respectively using DNAStar by row, search out and are suitble to design specific primer
Conservative region, then Primer Premier 5.0 is used to be analyzed and screened in conjunction with Oligo7.0, designed and by testing
Card filters out 2 pairs of specific primers (being shown in Table 1) expanded for H9 and H10 hypotype AIVHA genes.
1 primer information of table
Embodiment2：The foundation and optimization of duplex RT-PCR detection architecture
The extraction of 2.1 cause of disease RNA/DNA is synthesized with reference to nucleic acid extraction kit operation instructions with cDNA in this research
The RNA and ILTV of used AIV, IBV, NDV and ARV and the DNA of MG are stripped, nucleic acid-templated with 33 μ after extracting
LRNA-free water dissolutions.RNA templates carry out the synthesis of cDNA, all nucleic acid with reference to the specification of precious biological reverse transcription reagent box
It is spare that template sets -30 DEG C of Cord bloods.
The foundation of 2.2 duplex RT-PCR reaction systems and optimization this method use 25 μ L reaction systems：2×PCR Mix 10
μ L, H9 and H10 hypotype AIV cDNA templates each 1 μ L, primer H9-F, H9-R, H10-F and H10-R (25pmol/ μ L) are separately added into
0.1-1.0 μ L, totally 10 gradients, each gradient are incremented by 0.1 μ L, carry out the optimization of two primer combined concentrations, finally use RNA-
Free complements to 25 μ L.Optimization is combined to annealing temperature and reaction time according to the effect of concentration experiment again simultaneously, finally
Determine this method best reaction system and condition.
The primer for preparing HA full-length genes in bibliography of 2.3 standard items is respectively with H9N2 and H10N3 strains cDNA
Template carries out the amplification of HA genes RT-PCR, obtains its overall length target fragment, and target fragment is connected respectively on carrier simultaneously
Company is sent to be sequenced.By the HA full length genes segment inserted with H9 and H10 hypotypes AIV and correct recombinant plasmid difference is sequenced
It is named as H9-T and H10-T.The plasmid containing H9-T and H10-T is extracted respectively with the plasmid extraction kit of commercialization, is used in combination
Trace dna detector is measured its concentration, calculates the corresponding copy number of sample according to correlation formula, while by H9-T
It is mixed with copy numbers such as H10-T plasmids, and the sample mixed is carried out to 10 times of doubling dilution, to obtain H9-T and H10-
T plasmid DNA concentrations are 5 × 109~5 × 101The standard items of copy/μ L.
The optimization of 2.4 duplex RT-PCR reaction conditions
We are to H9 and H10 hypotype AIV HA genes two to the excellent of specific primer concentration ratio and amplification temperature-time etc.
Change, determines that the Optimal system of duplex RT-PCR reaction is：2 × PCR Mix, 12.5 μ L, H9 and H10 hypotypes AIV totally 2 μ L as mould
The addition of plate, specific primer H9-F and H9-R (25pmol/ μ L) is 0.75 μ L, specific primer H10-F and H10-R
The addition of (25pmol/ μ L) is 1 μ L, and final volume is complemented to 25 μ L with RNA-free water.By carrying out ladder to annealing temperature
Optimal screening is spent, determines that the optimum annealing temperature of the reaction system is 53 DEG C.
Embodiment3：Specific detection
With above-mentioned established duplex RT-PCR detection method according to H1N7, H1N6, H2N3, H3N2, H3N6,
H3N8、H5N1、H5N2、H5N7、H5N9、H6N1、H6N2、H6N5、H6N8、H7N2、H7N9、H8N4、H9N2、H9N6、H10N3、
The cDNA/DNA of H12N5, H13N5, H14N1, H15N5, NDV, ARV, IBV and ILTV are detected, the spy of verification institute construction method
It is anisotropic.
As shown in Figure 1, detecting 2 respectively from H9 and H10 hypotype AIV mixing samples with the duplex RT-PCR method established
The band of specificity, respectively 490bp (H9 hypotypes) and 272bp (H10 hypotypes)；H9N2 and H9N6 hypotype AIV PCR are detected
Result only there is 1 specific band, clip size 490bp；H10N2 hypotypes AIV is expanded and also only detects 1
Specific band, clip size 272bp；Any is not amplified to other hypotype AIV and common poultry diease pathogen
Band, the results showed that this method has good specificity.
Embodiment 4：Sensitivity Detection
With the good duplex RT-PCR detection method of above-mentioned optimization to prepare a concentration of 5 × 109To 5 × 101Copy/μ
H9-T and the H10-T Plasmid samples of L are expanded, and the sensibility of the detection method is verified.
As shown in Fig. 2, being directed to 5 × 10 with this method9-5×101The AIV plasmid templates of H9 and the H10 hypotype of copy/μ L
It is expanded, the results show that a concentration of 5 × 109-5×104The AIV of H9 and the H10 hypotype of copy/μ L have 2 it is apparent
Specific amplification band occurs, and clip size is respectively 490bp and 272bp；5 × 10 are equal to or less than to concentration3Copy/μ L's
H9 and H10 hypotypes AIV is without amplified band.It can be seen that the method most low energy detects that plasmid template amount is 5 × 104Copy/μ L
H9 and H10 hypotypes AIV.
Embodiment 5：Clinical sample detects
The duplex PCR detection method established with this experiment swallows the 150 portions of chickens acquired in the recent period from live-bird market and duck
Larynx and a cloacal swabs sample part carry out PCR Testing and appraisals, while identical number remaining sample being connect after treatment
Kind SPF chicken embryos carry out the separation and identification of influenza virus, and will identify the sample progress HA bases that H9 and H10 hypotypes AIV is positive
The measurement of cause.Then the result of above two method is compared, and then verifies the accuracy of duplex RT-PCR testing result.
As shown in figure 3, with the method established to respectively from the collected 120 portions of chickens in In Nanning Area difference live-bird market and
Duck throat and cloacal swab sample carry out PCR detections, and it is H9 and H10 hypotypes AIV that the result of sample detection, which shows 6 parts of samples,
Mixed infection is positive, and positive rate 5%, 21 parts of samples can amplify the purpose band of 490bp, is H9Nx AIV, 2 parts of sample energy
The purpose band of 272bp is amplified, is H10Nx AIV.The Virus Isolation and its HA genes of above-mentioned testing result and sample
Sequencing result 100% is consistent.
The description of the aforementioned specific exemplary embodiment to the present invention is in order to illustrate and illustration purpose.These descriptions
It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to the above instruction, can much be changed
And variation.The purpose of selecting and describing the exemplary embodiment is that explaining the specific principle of the present invention and its actually answering
With so that those skilled in the art can realize and utilize the present invention a variety of different exemplary implementation schemes and
Various chooses and changes.The scope of the present invention is intended to be limited by claims and its equivalents.
SEQUENCE LISTING
<110>Veterinary Institute of Guangxi Zhuang Autonomous Region
<120>Differentiate H9 and H10 subtype avian influenza virus duplex RT-PCR detection primers group, kit and its application simultaneously
<130> ZYWS
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
gcagcagata gagactcaac c 21
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence
<400> 2
caagagatga ggcgacagt 19
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence
<400> 3
acgaacactt acagaaacac gga 23
<210> 4
<211> 19
<212> DNA
<213>Artificial sequence
<400> 4
ctcggtgcta tcagacccc 19
Claims (5)
1. a kind of differentiating H9 and H10 subtype avian influenza virus duplex RT-PCR detection primer groups simultaneously, which is characterized in that including 2
To specific primer, it is primer pair H9-F and H9-R, primer pair H10-F and H10-R respectively, is respectively provided with such as SEQ ID
Sequence shown in No.1 to SEQ ID No.4.
2. a kind of differentiating H9 and H10 subtype avian influenza virus duplex RT-PCR detection kits simultaneously, which is characterized in that including：2
Totally 2 μ L are 12.5 μ L, H9 and H10 hypotypes AIV of × PCR Mix as template, the addition of specific primer H9-F and H9-R
The addition of 0.75 μ L, specific primer H10-F and H10-R are 1 μ L, and final volume is complemented to 25 μ L with RNA-free water.
3. it is according to claim 2 while differentiating H9 and H10 subtype avian influenza virus duplex RT-PCR detection kits,
It is characterized in that, the specific primer H9-F and H9-R, specific primer H10-F and H10-R are respectively provided with such as SEQ ID
Sequence shown in No.1 to SEQ ID No.4.
4. it is according to claim 2 while differentiating H9 and H10 subtype avian influenza virus duplex RT-PCR detection kits,
It is characterized in that, the specific primer H9-F and H9-R, specific primer H10-F and H10-R are corresponding in PCR reaction systems
A mole final concentration be respectively 0.75pmol/ μ L, 0.75pmol/ μ L, 1pmol/ μ L, 1pmol/ μ L.
5. it is according to claim 1 at the same differentiate H9 and H10 subtype avian influenza virus duplex RT-PCR detection primer group or
Differentiate that H9 and H10 subtype avian influenza virus duplex RT-PCR detection kits are differentiating while claim 2-4 is any described
Application in H9 and H10 subtype avian influenza virus.
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| CN111518954A (en) * | 2020-05-07 | 2020-08-11 | 广西壮族自治区兽医研究所 | Primer group, kit and method for double nano PCR detection of H5 and N8 subtype avian influenza virus |
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| CN110846437A (en) * | 2019-10-31 | 2020-02-28 | 广西壮族自治区兽医研究所 | Double PCR (polymerase chain reaction) detection primer group, kit and method for chicken parvovirus and H9 subtype avian influenza virus |
| CN111518954A (en) * | 2020-05-07 | 2020-08-11 | 广西壮族自治区兽医研究所 | Primer group, kit and method for double nano PCR detection of H5 and N8 subtype avian influenza virus |
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